The Ras family of GTPases are known to be critical in the transient activation of proliferative signals by extracellular mitogens. Oncogenic Ras proteins provide a constitutive, rather than transient signal, thereby resulting in cell transformation. Oncogenic Ras proteins can, in some instances, protect cells from apoptosis, primarily through the constitutive activation of PI-3 kinase and its downstream target, Akt. In other cases, oncogenic Ras can activate the apoptotic pathway, resulting in cell death. Using N-Ras and K(i)-Ras knockout cell lines, we have demonstrated that these proteins play a critical role in regulating apoptotic sensitivity in normal cells. In the absence of c-N-Ras expression, cells become hypersensitive to apoptotic agents, inclusive of TNFalpha, anti-Fas and serum-withdrawal. Ectopic expression of c-N-Ras in the N-Ras deficient cell lines restored their resistance to each of the apoptotic treatments. Ectopic expression of c-K(A)-Ras, however, failed to rescue the apoptotic sensitivity of the N-Ras knockout cell lines. In fact, expression of the K(A)-Ras protein enhanced even further the apoptotic sensitivity of the N-Ras knockout cell lines. Our preliminary data suggest that c-N-Ras deficiency results in a higher than normal level of mitochondrial instability. Raf-1, MEK1 and c-N-Ras are all found in a purified mitochondrial preparation, supporting our hypothesis that c-N-Ras plays a vital role in maintaining mitochondrial integrity. While N-Ras negative cells are hypersensitive to the induction of apoptosis, K(i)-Ras negative cells were more resistant than their cognate control cells (K+/+) to apoptotic agents, suggesting that cellular K(i)-Ras proteins provide a steady-state pro-apoptotic signal. We will test the hypothesis that endogenous cellular Ras isoforms (N, K(A), and K(B)-Ras) regulate the threshold that determines whether or not extracellular stimuli promote programmed cell death. We will test this hypothesis through the execution of the following specific aims: 1. We will determine which well-defined apoptotic components are altered by the lack o c-N-Ras expression. 2. We will characterize the requirement for cellular K-Ras proteins in sensitizing fibroblasts towards apoptotic signals. 3. We will perform structure/function analysis on the variable regions between N-Ras K(A)-Ras and K(B)-Ras to determine specific regions within these proteins which define their opposing functions in regulating cellular sensitivity towards apoptotic agents.